中国林蛙核糖核酸酶Rdrlec的原核表达及抗菌活性检测

Prokaryotic Expression and Antimicrobial Activity of a Novel Ribonuclease Rdrlec from Rana dybowskii

Rdrlec是从中国林蛙基因组中克隆得到的核糖核酸酶新基因(GenBank登录号EU384704),其生物学活性仍未知。为了探索其编码的蛋白产物的生物学活性,将此基因按照大肠杆菌偏好的密码子进行优化后,人工合成基因,通过EcoR I和HindIII限制酶位点定向克隆到pET-28a(+)中构建重组表达载体,转化到大肠杆菌BL21(DE3)中,在34℃以0.5 mmol/L IPTG诱导表达出包涵体形式的融合蛋白,通过包涵体复性、肠激酶切割、Ni-NTA亲和层析纯化等步骤获得了具有酶活性的野生型Rdrlec重组蛋白,并检测到其对大肠杆菌和金黄色葡萄球菌具有显著的抗菌活性。

Rdrlec is a novel RNase gene form Rana dybowskii（GenBank accession No.EU384704）and its biological function has not been identified.In order to explore the biological activity of its encoded protein,the Rdrlec gene was adjusted according to Escherichia colicodon bias without changing its amino acids.The synthetic gene was inserted to the pET-28a（＋）expression vector through the EcoR I and Hind III site,and the resulting recombination expression plasmid was named pET28-Rdrlec and transferred into Escherichia coli BL21（DE3）strains.After induced with 0.5 mmol/L IPTG at 34℃ for 6 h,the fusion protein was found expressed mainly in inclusion body form.After a series of steps including refolding,enterokinase cutting and Ni-NTA affinity chromatographic purification,the wild type Rdrlec recombinant protein was obtained,and it showed a single band on SDS-PAGE.It showed enzymatic activity to degrade RNA into nucleotides,which suggests that this molecule has formed the correct spatial structure.The recombinant Rdrlec shows significant antibacterial activity against Escherichia coli and Staphylococcus aureus.